Polysaccharides, such as starches, are complex carbohydrates composed of monosaccharides joined via glycosidic bonds. They are typically amorphous and insoluble in water. Examples of polysaccharides include storage polysaccharides such as starch and glycogen, and structural polysaccharides such as cellulose and chitin.
Starch is a major carbohydrate reserve in plant tubers and seed endosperm. The largest source of starch is corn (maize) with other commonly used sources including wheat, potato and rice. Starchy substances constitute a major part of the diet of humans in many parts of the world, as well as the diet of many animals. Starch, however, is important not only as a food and feed source, but also as an energy source, as in crop-based biofuels.
Given the increasing demands for food, feed and fuels, crops are being genetically modified to increase starch concentration and utilization. Consequently, genetic modifications of starch crops include development of starches with improved and targeted functionality. See, e.g., Jobling (2004) Curr. Opin. Plant Biol. 7:210-218. Other genetic modifications of starch crops include development of crops with increased starch-hydrolyzing capabilities. See, e.g., US Patent Application Publications No. 2006/0230473 or 2003/0135885 for example.
In commercial applications, starch is commonly converted to glucose and/or other simple sugars. The steps in converting starch to glucose are gelatinization, liquefaction and saccharification. Briefly, gelatinization is a swelling of starch granules by heat and water. During gelatinization, starch loses its crystallinity and becomes an amorphous gel that can be more easily accessed by hydrolyzing enzymes. Liquefaction is the hydrolysis of starch to dextrins by a hydrolyzing enzyme such as amylase. Similarly, saccharification is a hydrolysis of dextrins to glucose by an enzyme such as glucoamylase.
Current methods for detecting and measuring transgenic plant material containing a polysaccharide-hydrolyzing enzyme are time consuming in that they require milling the plant material, extracting the polysaccharide-hydrolyzing enzyme from a plant sample, adding exogenous polysaccharide as a substrate and/or hydrolyzing the polysaccharide for lengthy periods of time. Efficient food, feed and fuel production methods, however, require that one be able to accurately and quickly assess the level of polysaccharide-hydrolyzing enzyme activity. For example, insufficient α-amylase activity in transgenic corn flour for ethanol production may result in poor ethanol yield in that too little enzyme is available to actively liquefy the starch. With an increasing need and use of starchy crops, including genetically modified crops, for food, feed and fuel, there is a need for rapid, portable and inexpensive methods to detect and measure polysaccharide-hydrolyzing enzyme activity or concentration. For instance, it would be highly beneficial to have a method that could be used directly at a dry grind ethanol plant receiving corn seed expressing a alpha-amylase (for example, as described in U.S. Patent Application Publication No. 2003/0135885A1) to quickly quantify amylase activity in a small sample of said corn seed to ensure an adequate dosage of alpha-amylase is being added to efficiently support downstream starch liquefaction processes.